Ligation‐based assay for variant typing without sequencing: Application to SARS‐CoV‐2 variants of concern

Abstract Background COVID‐19 prevalence has remained high throughout the pandemic with intermittent surges, due largely to the emergence of genetic variants, demonstrating the need for more accessible sequencing technologies for strain typing. Methods A ligation‐based typing assay was developed to detect known variants of severe acute respiratory syndrome virus 2 (SARS‐CoV‐2) by identifying the presence of characteristic single‐nucleotide polymorphisms (SNPs). General principles for extending the strategy to new variants and alternate diseases with SNPs of interest are described. Of note, this strategy leverages commercially available reagents for assay preparation, as well as standard real‐time polymerase chain reaction (PCR) instrumentation for assay performance. Results The assay demonstrated a combined sensitivity and specificity of 96.6% and 99.5%, respectively, for the classification of 88 clinical samples of the Alpha, Delta, and Omicron variants relative to the gold standard of viral genome sequencing. It achieved an average limit of detection of 7.4 × 104 genome copies/mL in contrived nasopharyngeal samples. The ligation‐based strategy performed robustly in the presence of additional polymorphisms in the targeted regions of interest as shown by the sequence alignment of clinical samples. Conclusions The assay demonstrates the potential for robust variant typing with performance comparable with next‐generation sequencing without the need for the time delays and resources required for sequencing. The reduced resource dependency and generalizability could expand access to variant classification information for pandemic surveillance.

diagnostic tests. [1][2][3][4][5][6] Real-time monitoring and tracking of the genetic progression are essential for informing dynamic adaptation to the changing needs required to counter the pandemic. The gold standard for the discovery of emerging variants and surveillance of existing variants is viral genome sequencing. [7][8][9][10] Although sequencing is necessary for discovering new variants, as a method to classify variants, the strategy is expensive and requires access to high-level technical expertise and sequencing equipment.
To address the greater need and demand for population-level SARS-CoV-2 variant surveillance, nucleic acid amplification tests (NAATs) such as reverse transcription polymerase chain reaction (RT-PCR) have been explored. [11][12][13][14][15][16][17][18] The PCR-based strategies differentiate among variants through the detection of unique, characteristic singlenucleotide polymorphisms (SNPs). Several approaches demonstrate the SNP-recognition capabilities inherent to PCR when primers or hydrolysis probes have single base mismatches that alter the quantification cycle (Cq) value at which amplification occurs for a target. 11,17 Since these variant detection platforms use common real-time RT-PCR practices, they are readily employable for use in most laboratoryequipped settings. However, their reliance on slight changes in the efficiency of primer/probe binding makes these approaches susceptible to false negatives and positives. 19 To enhance the specificity of RT-PCR-based approaches, the implementation of minor groove binding hydrolysis probes 13 and locked nucleic acids 14 have been proposed and evaluated. These approaches demonstrated high clinical performance for the majority of samples of Alpha, Beta, Gamma, and Delta variants but were limited to samples with high viral titers (e.g., Cq values ≤ 30 cycles). Melt-analysis has also been coupled with RT-PCR to determine the presence of SNPs by recognition of mismatch melt temperatures. 12,16 Although these studies demonstrated promising results, external quality assessments of the melt-analysis and mutation-specific PCR assays often rely on the detection of multiple variant sites and require relatively high viral load for accurate results. 19,20 Additionally, their performance is negatively impacted when additional polymorphisms are present. 19,20 For sequence-free variant identification, we employ a ligationbased molecular approach that classifies variants based on characteristic SNPs. The oligonucleotide ligation assay (OLA) is a highly specific nucleic acid hybridization-based assay for the determination of SNPs. 21 The assay discriminates single bases by joining two directly neighboring target recognition ligation probes if and only if the probes hybridize to the target at the bases nearest the junction. The OLA characteristically performs with high specificity and, when paired with NAATs, such as PCR, can achieve high sensitivity to detect low abundance SNPs in RNA and DNA targets. [22][23][24][25][26][27][28] In this work, we present a generalizable method for the detection of known and future SARS-CoV-2 variants. The assay couples the OLA with a pre-amplification RT-PCR step and an endpoint real-time PCR detection method to recognize characteristic SNPs for current VOCs. The analytical and clinical performances of the assay were evaluated to demonstrate its applicability to real-world, population-level testing. The general principles used in the development of the assay suggest that the method can be readily adapted for future emergent VOCs as well as other diseases. Overall, the work here yields a generalizable method for population-level monitoring of known SARS-CoV-2 variants in settings where sequencing is either inaccessible or overwhelmed by high case numbers.

| Assay overview
The variant typing assay is designed to be generalizable for genetic surveillance of SNP patterns with clinical relevance. The assay sequentially links three enzymatic reactions for the amplification, discrimination, and detection of variant-characteristic SNPs (Figure 1).
Following RNA extraction, the assay is initiated by RT-PCR to amplify regions of interest (ROIs) that contain the characteristic SNPs indicative of variant type or other clinically relevant features. The amplified ROIs are transferred to specific subsequent OLA reactions to investigate the sequences containing the characteristic SNPs.
The OLA discriminates between nucleotides through a hybridization-based ligation event. The ligation occurs if and only if the 3 0 end of a synthetic ligation probe hybridizes to the single nucleotide of interest. By introducing multiple oligonucleotide ligation probes with alternate 3 0 nucleotides, known as variable probes (VPs), the ligation event, which occurs with a singular, always-binding common probe (CP), achieves very high specificity. Since the ligation F I G U R E 1 The ligation-based genetic variant typing assay is composed of three sequentially conducted enzymatic assays. Regions of interest (ROIs) that contain single-nucleotide markers of variants are first amplified by reverse transcription polymerase chain reaction (RT-PCR). A portion of the RT-PCR product is transferred to an oligonucleotide ligation assay (OLA) reaction where synthetic ligation probes are ligated into a single target if and only if hybridization occurs at the nucleotide of interest. A portion of the OLA product is then transferred to a real-time PCR reaction for endpoint detection of ligated strands. The PCR signal indicates the presence of particular SNPs of interest.
probes are synthetically manufactured, specific and exogenous primer and hydrolysis probe binding sites can be concatenated to the ligation probes for real-time PCR detection in the following reaction.
PCR is used to sensitively and specifically amplify transferred ligation product as a marker of the presence of SNPs of interest. Endpoint PCR was selected as the detection modality since a thermal cycler was already a pre-requisite of the assay for pre-OLA amplification, the platform is highly specific, and the additional amplification step provides extra sensitivity to aid in the detection of low viral load samples ( Figure S1). The PCR reaction employs exogenous primers and probes to enhance the specificity of the assay. Signals from each PCR reaction are used to determine the sample variant type in combination according to an SNP-based typing chart ( Figure 2).
In this SARS-CoV-2-specific implementation of the variant detection assay, there are two ROIs that are amplified from the receptor binding domain (RBD) within the spike gene. The RBD was selected because of the abundance of genetic mutations in each of the VOCs and its likely continued accumulation of mutations in future VOCs.
The two ROIs encapsulate (1) amino acid (AA) 339 and (2) AAs 452 and 484. These regions contain mutations unique to Omicron (G339D), Delta (L452R), and Beta/Gamma (E484K) as well as a marker for a positive control indicative of the wild-type sequence (E484). At the time of assay development, L452R was unique to Delta; however, the emergent sublineages of Omicron, BA.4 and BA.5, contain this SNP. In these cases, the assay differentiates between Delta and Omicron variants as BA.4 and BA.5 contain the G339D marker such that proper classification requires both signals. The SNP-based variant typing chart for this application is presented in Figure 2. In this work, two workflows were conducted in the evaluation of the assay. The first workflow tests for different SNP markers by performing each enzymatic reaction in a singleplex, whereas the second workflow combines each enzymatic reaction into a multiplex workflow ( Figure S0). The first workflow is presented to demonstrate the most generalizable approach to adapting this method, and the second workflow demonstrates the multiplexable nature of the assay to reduce the technical burden of assay performance.

| Design/rules for OLA-based genetic typing
The variant detection assay is generalizable for genetic typing of variant sublineages, future emerging variants, and for other diseases that can be classified by SNP patterns. The key components of the variant detection assay design process can be described in four primary steps:  (2) Creation of the common and variable ligation probe, CP and VP respectively, hybridization region by complementing the target strand in the sense that will be present in the OLA reaction. The length of each flanking region is determined by accounting for the desired melt temperature.
(3) Investigation of the hybridization or cross-reactivity of the exogenous primers and hydrolysis probes that will be used for downstream real-time detection as well as the ligation probe flanking regions that will be in the OLA reaction. (4) Concatenation of the primer and flanking regions for each ligation probe as well as the hydrolysis probe binding region for the SNP-discriminatory VP.
identified by existing known sequences. As sequencing data are collected, consensus sequences are generated and the one or more consistent mutations that differentiate an emerging variant from others are determined based on a set of determined nomenclature rules. 29 To employ this assay, it is assumed that new variants are classified prior to selecting the SNPs of interest. The mutations that were used to classify new variants are the characteristic SNPs that should be targeted in this assay design. As the target of interest, in this case, SARS-CoV-2, evolves, some SNPs may be signaled in other variants. In this study, the Delta discriminating SNP evolved in later sublineages of Omicron. These cases rely on information from multiple channels to differentiate with continued success (e.g., G339D positive or negative signal may distinguish Delta from Omicron). Also, multiple SNPs may be present in the sequences for differentiation, in which case one is selected and evaluated for its simulated performance, described in Step 3, and its experimental performance. Reselection of the SNP of interest may be necessary following the results of the proceeding steps. An example of a ligation set that needed to be reselected is shown in Figure S2. However, many additional tools aid in the characteristic SNP identification process; some of which provide lists of variant-specific single-nucleotide mutations that are useful for ready adaptation of the ligation-based assay presented here. [30][31][32][33] Once the single-nucleotide mutation of interest is selected, the target-hybridizing regions of the ligation probes are designed  36 In this design, the HiFi Taq Ligase is utilized, which requires the VP to hybridize with the sequence downstream of the SNP of interest. However, other ligases enable the VP to hybridize either upstream or downstream of the mutation, which may affect the fidelity of the SNP discrimination. Additionally, alternative ligase enzymes may have varied dependence on other nonsynonymous mutations near the SNP of interest that alters the enzyme efficiency and specificity. 35 If alternative ligases are desirable for others' intended use, these effects should be considered in the assay design.
For the ligation-based variant assay to yield the greatest specificity, detection of the ligated product by real-time PCR should be achieved using primers and hydrolysis probes with sequences that are not likely to be found in the clinical sample. In this work, primers and probes were selected for infectious diseases that are unlikely to coinfect a COVID-19 patient in the region of our study as these PCR reactions had been developed for previous projects (e.g., Zika Virus).
In practice, the background target is not likely to cause a false signal because of the employment of highly specific sequential reactions.
Alternative schemes that intentionally mismatch the primer and probe targets may also be used in this modular assay design. The highly spe-  Samples were added to spin columns at a volume of 140 μl and eluted in a final volume of 60 μl of Qiagen AVE buffer.

| Variant assay
The variant detection assay is intended for use with known COVID-19-positive samples. It was designed to detect four major VOCs that have emerged throughout the course of the pandemic: Alpha to circulate, and thus, the assay was designed to combine these two variants into a single output. The variant assay workflow was initiated with pre-amplification on extracted samples by RT-PCR. Product from the RT-PCR reaction was directly pipetted into the OLA for SNP discrimination. Detection of the ligated targets was performed by realtime PCR following direct pipette transfer of a fraction of the OLA F I G U R E 4 Ligation-based variant typing produced a highly specific signal for the detection of unique single-nucleotide polymorphisms (SNPs) in synthetic SARS-CoV-2 DNA targets or RT-PCR products of purified genomic RNA from BEI resources. (A) The Alpha and Beta singlenucleotide markers, E484 (yellow) and E484K (green), are performed in a single multiplexed OLA reaction without cross-reactivity for the detection of 10 6 copies of synthetic DNA targets. The multiplexed E484(K) ligation set amplifies in all COVID-19-positive samples and, therefore, serves as a sample control for the assay. (B) Singleplex OLA for the L452R marker (red), characteristic of the Delta variant, specifically detects the mutation without non-specific amplification of the wild-type L452 marker (blue) for the RT-PCR product of Delta variant purified genomic RNA. (C) The G339D mutation (orange) is detected as a characteristic marker of the Omicron variant. The ligation reaction for G339D does not produce amplified signal for the wild-type G339 marker (purple). The G339D ligation pair was evaluated on 10 6 copies of synthetic DNA target. The representative data show desired performance of ligation reactions in the screening process. product (Figure 1). Each step of the assay is outlined in the following methods section. All synthetic oligonucleotides including primers, hydrolysis probes, and ligation probes were synthesized by IDT. The composition of each reaction in the variant typing assay is tabulated for reproducibility in the Supporting Information (Tables S1-S3).  Table 1. Pre-amplified targets were transferred to the subsequent reaction immediately or stored at 4 C until ready for use.

| Oligonucleotide ligation assay
Following the pre-amplification step, 10% (2 μl) of the RT-PCR product was transferred into its SNP-specific OLA reaction. Three SNPspecific OLA reactions targeting E484/E484K, L452R, and G339D were employed for the detection of the four variant groups aforementioned. The RT-PCR product encompassing AA339 was added to the G339D OLA reaction, and the product containing AA452 and to reduce cross-over noise that was occurring in the reaction. This cross-over reduction was observed by empirical study (data not shown). Molecular-grade water was added to bring the reaction to 20 μl total. The reaction was performed with a 95 C hold for 1 min followed by a 60 C hold for 10 min. The ligation probe sequences are reported in Table 1.

| Multiplex variant assay
The variant assay was also converted to a multiplexed format to enhance the ease of assay performance. Pre-amplification RT-PCR was conducted by including both primer sets for the two ROIs. Each   To ensure the robustness of the study, the viral load distribution of the clinical samples investigated in this study was compared with the population-level viral load distribution from samples collected at VUMC from March 2020 to March 2021 (n = 5160) ( Figure 6). The study sample viral load distribution was not found to be statistically different from the population viral load distribution.

| Multiplex assay performance
Following singleplex clinical trials, multiplexing of the variant assay was investigated to reduce the number of assay steps required to conduct the assay. In the multiplexed format, the three sequential enzy-  (Table S5). The multiplexed format demonstrated an overall concordance with a singleplex of 98.6%, accounting for all true positives and negatives based on the singleplex reaction as the ground truth.
Compared with NGS, the multiplexed format had an overall clinical sensitivity and specificity of 93.2% (CI: 85.7%-97.5%) and 99.0% (CI: 96.4%-99.9%), respectively (Table 4).  Note: The multiplexed assay was performed on the same extracted nasopharyngeal COVID-19 patient samples as singleplex testing. Sample identification accuracy was determined by comparison with next-generation sequencing. Concordance was determined by the summation of true positive and negatives divided by the total number of samples in which singleplex results were considered the ground truth (n = 88 positive COVID-19 samples, 7 independent trials).

| Ligation performance with polymorphisms in the hybridization region
A major benefit of the demonstrated approach is the generalizability of the assay. In this work, a guide to reproducing and extending the principles of the ligation-based variant typing assay is provided.
Additionally, a design flow chart is provided in the Supporting The current iteration of the ligation-based variant typing assay could be enhanced by the inclusion of automated processing. The assay requires manual processing to transfer products through the sequential enzymatic reactions, which can be burdensome on the operator and increase the risk of laboratory contamination from opening tubes containing RT-PCR amplicons. Future automation by strategies such as magnetic processing or robotic liquid handling through a self-contained platform would minimize contamination and technical burden. [44][45][46] Optimizing the workflow to enable testing of unextracted samples would further improve the processing burden. 39 The ligation-based assay may also benefit from adopting alternative amplification and detection strategies that do not require a real-time PCR instrument. However, in its current state, since the assay employs real-time PCR for endpoint detection of variants, the utilization of the RT-PCR as the pre-amplification enables operators to use the same equipment. Additionally, the high sensitivity of the endpoint PCR step enhances the capabilities of the assay to detect low viral load samples. Yet, overcoming the need for a PCR device would further improve the accessibility for the generalizable genetic-typing assay.
Overall, the work presented demonstrates a sequencer-free genetic variant typing assay that robustly detects known variants of SARS-CoV-2 in a manner that is sustainable through the evolution of the virus. The ligation-based assay is readily adaptable to future SARS-CoV-2 variants and to other diseases for the detection of F I G U R E 7 The ligation-based variant detection assay performed robustly in the presence of surrounding non-synonymous single-nucleotide polymorphisms (SNPs). The Delta and Omicron variants of concern developed SNPs in the ligation probe hybridization region of the ligation control/Alpha detection ligation set (left). In clinical samples, the presence of a single polymorphism in the hybridization region, found in the Delta samples, did not significantly alter the performance of the assay (right). The presence of multiple polymorphisms, most importantly E484A which occurs at the base immediately neighboring the base affected by the E484(K) mutation, as found in the Omicron clinical samples, resulted in a significant delay in endpoint PCR detection (paired two-sample t-tests with Bonferroni correction for four groups, α = 0.05. *p < 0.05, **p < 0.025, ***p < 0.01).
characteristic SNPs or other single bases of interest. Although the approach presented in this work cannot replace the discovery capabilities of genetic sequencing, the variant typing assay provides a promising means for expanding access to SARS-CoV-2 variant monitoring and detection.

ACKNOWLEDGMENTS
We would like to thank and acknowledge Mackenzie Attwell, Tim Williams, and Erin Reid for their contributions of sample management and processing.

CONFLICTS OF INTEREST
There are no conflicts of interest to declare.

ETHICS STATEMENT
All specimens were collected, de-identified, and transferred from Vanderbilt University Medical Center as approved by the Institutional Review Board (IRB #201708 and #201804).

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1111/irv.13083.

DATA AVAILABILITY STATEMENT
Data associated with this study is available in the main text and Supplementary Information. Additional data, including raw PCR data files and sequence FASTA files, will be made available at 10.5281/zenodo. 7036209 for reviewers and to the public following publication.